THE ROLE OF IMMUNE EFFECTOR CELLS IN THE 4T1.2 MURINE MAMMARY TUMOR MODEL
Open Access
- Author:
- Duan, Shizhao
- Graduate Program:
- Nutritional Sciences
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- November 14, 2018
- Committee Members:
- Connie Jo Rogers, Thesis Advisor/Co-Advisor
- Keywords:
- Breast Cancer
immunotherapy
preclinical breast cancer
triple-negative breast cancer
murine model
4T1.2 - Abstract:
- ABSTRACT Breast cancer is the most commonly diagnosed cancer among women and the second leading cause of cancer related death in the US (1). Despite recent advances made in breast cancer research, triple negative breast cancer (TNBC) remains the subtype with the poorest prognosis due to limited treatment options. The 4T1 murine mammary metastatic model has been widely used as a model for TNBC, because of the similarities in hormone receptor profile to human TNBC (2). One caveat of the 4T1 model is that there are few known tumor associated antigens which has limited the use of this model in preclinical immunotherapeutic studies (3, 4). The firefly luciferase gene, luc2, has been transfected into various tumor cell lines as a reporter gene for imaging studies. However, previous studies demonstrate that the luciferase epitope can induce CD8+ cytotoxic T cell specific responses, thus may be serving as a tumor antigen for immune recognition (5-7). The goal of the current study was to explore the role of luciferase in 4T1.2 cells, a clone that was selected for greater metastatic potential, and a model used to evaluate nutrition and exercise interventions in our laboratory. Specifically, we compared in vitro and in vivo growth rates, the role of immune components in tumor growth, metastatic progression and survival rate in the parental 4T1.2 and luciferase transfected 4T1.2 clone (4T1.2luc). The goal of the first study was to determine if luciferase expression by the 4T1.2luc cell line altered the in vitro proliferative capacity compared to the parental 4T1.2 cell line. We cultured both 4T1.2 and 4T1.2luc mammary tumor cell lines (2,500 cells/well in serial dilution) in triplicate for 72 hours, and assessed in vitro proliferation using the MTS assay. The proliferation rate of 4T1.2luc cell line did not differ significantly from 4T1.2 cell line, which indicated luciferase expressed by the 4T1.2luc cell line is not actively involved in signaling cascades mediating cell proliferation. In the second study, we examined in vivo growth rates of 4T1.2 and 4T1.2luc cells in immunocompetent BALB/c mice. We demonstrated that 4T1.2luc tumor-bearing mice had a significantly reduced primary tumor growth compared to 4T1.2 tumor-bearing mice. Next, we wanted to determine which immune subtype was responsible for the delayed tumor growth observed in 4T1.2luc tumor-bearing mice, and if any of these immune population were important in controlling metastatic progression or survival. To this end, we depleted the major effector immune cell populations, CD4+ T cells, CD8+ T cells and NK cells, in both 4T1.2 and 4T1.2luc tumor-bearing animals. We observed that CD4+ T cell, CD8+ T cell and NK cell depletion did not affect primary tumor growth in 4T1.2 tumor-bearing mice. However, CD8+ T cell depletion increased primary tumor growth and NK cell depletion decreased primary tumor growth in 4T1.2luc tumor-bearing mice. Metastatic burden in the lung and femur was not significantly different among isotype control, CD4+ T cell-depleted and CD8+ T cell-depleted, and NK cell-depleted 4T1.2luc tumor-bearing mice. The final aim of this study was to determine the role of CD4+ T cells, CD8+ T cells and NK cells in the survival of 4T1.2 and 4T1.2luc tumor-bearing mice. We observed that 4T1.2luc tumor-bearing mice treated with PBS or the isotype control antibody (LTF-2) had a significantly longer median survival than PBS or isotype control antibody-treated 4T1.2 tumor-bearing mice. In 4T1.2 tumor-bearing mice, CD4+ T cell, CD8+ T cell and NK cell depletion did not alter median survival. However, CD4+ T cell, CD8+ T cell depletion reduced median survival in 4T1.2luc tumor-bearing mice in comparison to control groups. In conclusion, luciferase expression in the 4T1.2luc cell line did not alter the in vitro proliferation rate but potentially served as an antigen for immune cell recognition. In 4T1.2luc tumor-bearing mice, CD8+ T cells are important in controlling primary tumor growth. Depletion of CD4+ T cells, CD8+ T cells and NK cells did not significantly affect lung and femur metastatic burden. However, both CD4+ T cell and CD8+ T cell populations are important for survival. In contrast, the absence of CD4+ T cells, CD8+ T cells or NK cells did not significantly influence primary tumor growth or survival in 4T1.2 model. Results from the current study provide important information about the interaction between host immune components and 4T1.2 and 4T1.2luc tumor cells.